Morphometric and Genetic Characterization of Dominant Fish Species in Progo River, Yogyakarta, Indonesia

. A study on the morphology and genetics of the three species of dominant wader that live in the Progo River has been conducted. Tis study aimed to identify the three kinds of dominant waders in the Progo River, their morphology, and their genetics to ensure their right identity. Fish characterization was done by morphological analysis using the truss-morphometric method and genetic analysis using molecular markers. Random amplifed polymorphic DNA (RAPD) based on PCR was used for diversity iden-tifcation for interspecies and between species. Several analyses of water quality parameters (temperature, nitrate, phosphate, and ammonia) were also carried out to determine the habitat’s suitability for the fsh’s survival. Based on the results of discriminant function analysis, all wader fsh from the Progo River used in this study were classifed into three species with the highest diferentiation character D4 (distance between the starting point of upper tail fn and the starting point of lower tail fn). Results also show that wader abang fsh have similar morphometric characteristics and DNA closer to wader kepe compared to wader pari . Te length-weight relationship of wader fsh from the Progo River shows that the growth pattern of wader abang and wader kepe is allometric positive, while that of wader pari is allometric negative. Te value of the condition factor for wader fsh from the Progo River indicates that the environmental habitat of wader fsh is good and stable so that it can support the fsh growth.


Introduction
Wader, a local term for fsh belonging to the family of Cyprinidae, is a dominant freshwater fsh species living in the Progo River (the longest river in Yogyakarta) [1].It has become a special raw material for favorite culinary foods in Yogyakarta.Fish is typically prepared in various ways, from simply fried in vegetable oil to cooking with many types of spices [2].People are eager to consume it due to its delicious and savory taste.Many restaurants in Yogyakarta feature wader dishes as their main menu.Wader is caught directly from its natural habitat [3].Tis exploitation has led to a continuous decline in the fsh population in the river [4].
To protect these species from extinction and explore possibilities for development (such as domestication and breeding), it is imperative to conduct a study to gather data, with a particular focus on morphological and genetic information.
Preliminary studies have revealed that the wader population in the Progo River comprises several species known locally as wader pari, kepe, melem, abang, bader, palung, and cakul.But the three dominant species of wader are wader abang, wader pari, and wader kepe.Although the three species have the same local name as wader, they have different morphological appearances.Wader abang is characterized by red fns, wader pari with blue-yellow stripes along their body, and wader kepe with yellowish fns and black stripes at the end of their fns.Wader abang is the biggest size among the three and wader pari is the smallest.Up to now, only those species mentioned above are known by the people even though there might be some more species of wader that live in the Progo River.Studies on taxonomical and species identifcation are still limited.Terefore, a study on the morphology and genetics of the three species of wader is necessary to confrm their correct classifcation.
Fish can be characterized by analyzing their morphology through the truss-morphometric method and their genetics using molecular markers such as random amplifed polymorphic DNA (RAPD) based on PCR [5,6].Tis study also analyzed condition factors and growth patterns (lengthweight relationships) to determine the characteristics of wader fsh based on their environment.

Sample Collection of Fish in Progo
River.Samples used in this study were various wader fsh species found in the middle area of the Progo River located around Nanggulan District, Kulon Progo, Special Region of Yogyakarta (7 °45′14.1″S110 °13′13.4″E)(Figure 1).Fish were caught using fsh nets by the local fshermen.Tirty fsh of various sizes were randomly sampled in fve locations, and their morphology was measured on the site.Te total and standard lengths of the fsh were measured using a ruler, and the weight was measured using a digital balance SF400C.Te fsh fns were taken and kept in 90% alcohol prior to DNA analysis.Water quality (temperature, pH, dissolved oxygen/ DO, phosphate, nitrate, and ammonia) was measured or analyzed at the same time as the sample collected.

Morphological
Analysis.Morphological analysis of wader fsh was conducted using truss-morphometric analysis [7].Each wader species with a complete body was selected as the fsh sample.Ten, truss points were selected based on [7].Te distance between specifc points on the fsh's body was measured by connecting certain points.Tirty fsh of each type were randomly selected from small to large sizes, marked with a needle, and measured using the truss-morphometric method.Distance measurements between points on the fsh's body were taken using a ruler.Each truss-morphometric character was divided by the fsh's standard length.Te fsh body was divided into 4 parts (A: head, B: front body, C: rear body, and D: tail) with 10 truss points ((1) pectoral fn, (2) mouth, (3) pelvic fn, (4) gills, (5) base of the anal fn, (6) base of a dorsal fn, (7) end of the anal fn, (8) end of the pectoral fn, (9) lower base of the anal fn, and (10) upper base of the anal fn).Te truss is connected, resulting in 21 morphometric truss characters that describe fsh diversity (Figure 2).Measurement results of the trussmorphometric were analyzed using discriminant function analysis (DFA) in SPSS version 25.

Molecular Analysis of Wader Fish Using the Method of
Random Amplifed Polymorphic DNA (RAPD).Molecular characterization of wader fsh was conducted using random amplifed polymorphic DNA (RAPD) analysis.DNA was extracted using the method of TIANamp Marines Animal DNA Kit 180123.RAPD analysis was conducted using primers OPA-01 and OPA-02.Te amplifcation process was performed using the method of polymerase chain reaction (PCR) RAPD with the composition of 3 µl DNA, 1.5 µl primer, 12.5 µl Taq DNA polymerase, and 8 µl distilled water.It was then put into a thermocycler with 1 denaturation cycle at 94 °C for 2 min, 40 doubling cycles consisting of denaturation at 94 °C for 1 min, annealing at 36 °C for 1 min, extension at 72 °C for 2.5 min, and fnal extension at 72 °C for 7 min.Te PCR results were analyzed through the process of electrophoresis, which involved using a 1.5% agarose gel in Tris-Boric EDTA (TBE) bufer at a voltage of 100 V for 30 minutes.Te marker used had a size range of 100-3000 bp and was made by Termo Scientifc.Te electrophoresis results were visualized under UV light after staining the agarose gel with FluoroVue nucleic acid gel stain and were documented using gel doc.
Te data analysis process was started by examining the presence of DNA fragments in the electrophoresis results of RAPD primers.Each fragment was considered a single locus and translated into binary data, where "1" indicated the presence of the fragment and "2" indicated its absence.TFPGA software was then used to analyze the binary data, generating a genetic distance value (D) [9].A smaller genetic distance value indicates a greater number of shared fragments between individuals, whereas a larger genetic distance value implies a greater diference in fragments.

Length-Weight Relationship Analysis and Condition
Factors of Wader Fish.Tirty fsh of various sizes from small to large were randomly sampled in fve locations within the Progo River, and their morphology, length, and weight were measured on the site.Te fsh's total length was measured from the mouth to tail using a ruler accurate to 0.5 mm in cm, and its weight was measured using a digital balance accurate to 0.01 g.Te length-weight relationship was calculated following the [10] method.
where W � total weight (g), L � total length (cm), a � intercept, and b � slope.Te growth pattern of fsh was determined from constant b (slope) calculated from length and weight through a hypothesis as follows: H0: if b � 3, the growth pattern is isometric (length growth equal to weight growth).H1: if b ≠ 3, the growth pattern is allometric, namely, (a) if b > 3, allometric positive (weight through more dominant).(b) If b < 3, allometric negative (length growth more dominant).Furthermore, the condition factors of fsh types were calculated using the condition factor equation of Fulton and relative weight.Te Fulton condition factor (K) was calculated using the Okgerman [11] equation as follows: where K f � Fulton condition factor, W � fsh weight (g), and L � fsh length (cm).Te relative weight condition factor of fsh was calculated using the equation of [12] as follows: where Wr � relative weight condition factor, W � fsh weight (g), and Ws � predicted fsh weight based on the linear allometric model (LAM).
Te results can then be compared with the results of other studies on the same fsh species or results from different habitats.

Water Quality Measurement.
As supporting data, condition factors of fsh and water quality (temperature, pH, dissolved oxygen, nitrate, ammonia (NH 3 ), and phosphate) of the sampling location were analyzed.Before analysis, the river water was taken using a bottle sampler.Te water quality was analyzed on-site, using a test kit.Ammonia was analyzed using Tetra Ammonia NH3.NH4 Test Kit, nitrate was analyzed using API Nitrate (NO 3 ) Test Kit, and phosphate was measured using a Compact Laboratory Water Tester.All three test kits use reagents in the process, which create color in proportion to the compound amount or concentration measured.Dissolved oxygen (DO) was measured using a Lutron D5509 DO meter, while pH was measured using a Kedida CT 6022 pH meter, and the temperature was measured using a mercury thermometer.Both DO and pH meters were calibrated before use.Te DO meter was calibrated following the calibration instruction manual, while the pH meter was calibrated using a pH bufer solution from pH 4.01, pH 6.86, and pH 9.18.Te water quality results were compared to government regulation no.22 of 2021 (water quality standards) in Appendix VI [13].

Te Morphological Character of Wader Fish.
Truss-morphometric analysis was performed covering 21 characteristics of three dominant wader fsh, i.e., wader abang, wader pari, and wader kepe.Te mean, standard deviation, and coefcient of variation of the tested parameters are presented in Table 1.
Te coefcient of variation of 21 morphometric characters of wader fsh ranges from 12.03 to 56.79%.Te character with the highest CV is C1 (distance from the base to the end of the anal fn), while the lowest is A4 (distance from the center between the head and the dorsal fn to the end of the pelvic fn) (Table 1).
Te analysis of morphometric data uses discriminant function resulting in 2 functions, i.e., functions 1 and 2 (Table 2).Discriminant function analysis was conducted to determine the contributing function of grouping morphometric data into diferent groups.Function 1 with an Eigenvalue of 9.272 explains 83.5% of the total variance, and function 2 with an Eigenvalue of 1.832 explains the rest of 16.5% of the total variance.All characters contributed to function 1, and character D4 (distance between the upper base and lower base of the anal fn) was the highest contributor (Table 2).
Figure 3 shows that functions 1 and 2 diferentiate wader fsh into 2 diferent groups.Function 1 is grouping wader fsh into 2 groups, i.e., wader pari on the left of 0 on the X axis (negative correlation) and wader abang as well as wader kepe on the right of 0 on the X axis (positive correlation).Function 2 also diferentiates wader fsh into 2 groups, i.e., one group above 0 on the Y axis (positive), i.e., wader abang, a small portion of wader kepe, and a portion of wader pari and one group below 0 on the Y axis (negative), i.e., wader kepe and part of wader pari.Tough they are diferentiated into diferent groups, wader abang and wader kepe still intersect each other.
Te classifcation result of the discriminant canonical function of wader fsh from the Progo River is presented in Table 3. Predicted group membership in the classifcation was based on the value of the degree of morphometric character similarity measured in the analysis [10].Te results of classifcation showed that 96.6% of the morphometric data of wader fsh from Progo River were able to be classifed.Te population of wader pari was completely separated (100%) from the other population, and those of wader abang and wader kepe were 92.6% and 97%, respectively.Tere was a morphometric character similarity of 7.4% between wader abang and wader kepe, and on the other hand, there was a morphometric character similarity of 3% between wader kepe and wader abang.

Te Genetic Character of Wader Fish from the Progo River
Is Based on the Method of Random Amplifed Polymorphic DNA (RAPD).Te analysis result of RAPD is presented in Figure 4. OPA-1 and OPA-2 primers have fragments that can be used as diferentiators among the tested wader fsh.Some RAPD fragments amplifed by OPA-1 and OPA-2 primers indicate that the three types of wader fsh are diferent species.Te three types of wader fsh have their specifc fragments, for example, amplifcation with OPA-1 primer results in 1200 and 1500 bp fragments which are found only in wader pari, 900 and 1100 bp fragments only found in wader kepe, or 700 bp fragments in wader abang.Te same thing is also seen in the RAPD fragment which was simplifed using the OPA-2 primer.Wader abang has a specifc fragment of 1600 and 790 bp, wader pari 800 and 1500 bp, and wader kepe 550 and 900 bp.
In general, the RAPD fragment of wader abang which was amplifed with OPA-1 and OPA-2 primers had many similarities with wader pari compared with wader kepe (Figure 4).Tis result can be seen in the genetic distance (Table 4).Based on genetic distance calculation, the three types of wader fsh living in the Progo River had a relatively far distance from each other.Te closest genetic distance was between wader abang and wader pari, i.e., 0.6439, followed by the genetic distance between wader abang and wader kepe, i.e., 0.7809, and the farthest was between wader pari and wader kepe, i.e., 0.7963.

Length-Weight Relationship and Condition Factor of
Wader Fish.Based on the length-weight relationship analysis of wader fsh, the b values of wader abang � 3.4311, wader kepe � 3.1618, and wader pari � 2.8705, so the equation of the length-weight relationship of the three fsh was as follows: wader abang was W � 0.0033L 3.4311 , wader kepe was W � 0.0068L 3.1618 , and wader pari was W � 0.0102L 2.8705 (Figure 5).Based on a t-test with a 95% confdence level, the growth patterns of wader abang and wader kepe were allometric positive, i.e., the weight growth rate was higher than the length growth rate.In contrast, the growth pattern of wader pari was allometric negative meaning that the length growth rate was higher than the weight growth rate (Table 5).
Te mean value of the condition factor of Fulton (K) for wader abang was 1.07 ± 0.17, while wader kepe was 0.02 ± 0.20 and wader pari was 0.77 ± 0.14 (Table 6).Tis result showed that wader abang had a round shape, while wader kepe and wader pari had a very fat shape.Wader abang had a relative weight condition factor (Wr) with a mean value of 99.47 ± 10.742, while wader kepe was 101.56 ± 19.590 and wader pari was 95.437 ± 25.266.Tis result showed that the surrounding water quality of wader fsh was relatively good.

Te Morphological and Genetic Characteristics of Wader
Fish in the Progo River.Wader fsh is one of the freshwater fsh types found in the Progo River which has a high economic value and is very potential to be developed.Tis fsh is caught or harvested directly from its natural habitat resulting in a continuously decreasing stock in the river.To protect this species from extinction, a study to obtain data and information about its biological condition needs to be conducted.Terefore, the preservation of the existence of wader fsh through domestication and breeding programs starts with collecting information and a genetic database of wader fsh living in the Progo River through analysis of morphological characteristics (truss-morphometric) and their genetic (RAPD).
Based on truss-morphometrics analysis, the coefcient of diversity (CV) and the characteristics of wader abang, wader kepe, and wader pari in the Progo River have high averages, i.e., ranging between 12.03 and 56.6%.Tis CV value is higher than the CV of the other fsh such as rice feld eel (3.01-14.65%)[14], tengadak fsh (2.24-12.76%)[15], kelabau padi fsh (6.89-10.77%)[16], nilem (3.38-10.35%)[17], and gourami 10.5% [18].Te diversity coefcient value of a character indicates the diversity level of a population [15].Reference [19] stated that morphological diversity is infuenced by some factors, namely, genetic factors inherited by the parent, adaptation of body form, color, and fn at the environmental condition in which they live, and adaptation Te Scientifc World Journal    Te Scientifc World Journal of head form for feed processing.Te high morphological diversity becomes a chance to increase fsh genetic variation.As mentioned by [20], the higher the CV value of a species, if there is crossbreeding among populations, the higher the chance of gene exchange among the populations.Discriminant function analysis on the morphometric data aims to determine if a character can be used to differentiate a species from a population which is very hard to diferentiate [12].In this study, discriminant function analysis was able to diferentiate wader fsh in the Progo River into three groups/populations.Wader abang, wader kepe, and wader pari form their character groups though there is an intersection of wader abang and wader kepe (Figure 2 and Table 4).Tis intersection shows that an individual has the same morphometric character between wader abang and wader kepe.Based on the classifcation result, there is a 7.4% similarity between wader abang and wader kepe and there is a 3% similarity between wader kepe and wader abang.In line with morphometric analysis, the results of RAPD analysis show that the three wader fsh from the Progo River have a long genetic distance, i.e., 0.6439-0.7890(Table 7).Tis result supports the assumption that the three-wader types in the Progo River are diferent species.Genetic distance can describe the pattern of kinship among populations.Te smaller the genetic distance (D), the closer the kinship between two populations and vice versa [21].Te genetic distance of wader fsh in this study is longer than that of ringau fsh (Datnioides microlepis), i.e., 0.1248 [22], giant snakehead (Channa micropeltes), i.e., 0.2471 [23], black and red nila (Oreochromis sp), i.e., 0.1414-0.6553[24], tengadak fsh (Barbonymus schwanenfeldii), i.e., 0.48-0.55[25], butini fsh (0.2114-0.6272) [26], and Malaysian river catfsh (Mystus nemurus), i.e., 0.390-0.635[27].Te genetic distance of the three wader fsh in this study is close to those of nilem hijau and nilem were or nilem beureum panon in West Java, which suggests that the three types of the nilem fsh are three diferent species [28].
Based on canonical function discriminant and classifcation results, wader abang fsh has a higher morphological similarity with wader kepe than wader pari.Tis result is in line with the result of RAPD analysis in which, based on molecular analysis, the genetic distance between wader abang and wader kepe is more than that of wader pari.It shows that, genetically, wader abang fsh has higher similarity with wader pari.Tis diference is suspected that wader kepe and wader abang have the same genera of Barbus, but wader pari belongs to the genera of Rasbora.On observation of color and body shape based on the existing references, wader abang has morphological similarity with balar/brek (Barbonymus ballaroides or Barbus bramoides) and wader kepe is similar to Barbus marginatus or Mystacoleucus marginatus, while wader pari has morphological similarity with Rasbora sp (Figure 6).

Length-Weight Relationship and Condition Factor of
Wader Fish.Length-weight relationship character of fsh becomes an important morphometry parameter in fsheries resource management [32][33][34].Tis information can be used as basic knowledge for explaining the diferences between the application of length size and standardization of length size in growth comparison studies [35,36].Tey are also used to compare diferent populations or fsh species [37].
Te length-weight relationship of wader fsh in the Progo River has been conducted to determine their growth pattern.Wader abang and wader kepe have an allometric growth pattern positive with a "b" value of 3.4311 and 3.1618, respectively, while wader pari has an allometric negative with a "b" value of 2.8705.Te diferences in the growth pattern of wader abang and wader kepe with wader pari are suspected due to the diference in activity that wader abang and wader kepe are passive swimmers, while wader pari is an active swimmer which needs much energy to move, so it has negative allometric growth.According to [38], active swimmer fsh have a b value relatively lower than passive swimmer fsh.Tis is related to the energy used for movement and growth [39].Other than movement, gonad development, feed availability, and body size variation have been reported to afect the b value [40,41].Te positive allometric growth pattern of wader abang and wader kepe in this study is similar to that of female Bader fsh (Barbonymus ballaroides) [42], lalawak fsh (B.ballaroides) which have been domesticated [43], wader bintik dua [44], bilih fsh (Mystaecoleucus padangensis) [45], and male gengehek fsh (M.marginatus) [46,47].Furthermore, the growth pattern of wader pari with the allometric negative is in line with the growth pattern of depik fsh (Rasbora tawarensis) [48] and wader pari fsh (R. argyrotaenia) [39,[49][50][51][52] as well as Rasbora fsh [53].
Te length-weight relationship chart (Figure 5) shows the value of the correlation coefcient (r) of wader abang fsh, wader kepe, and wader pari which are close to 1 (0.852-0.984).It shows that there is a strong correlation between the length and weight of wader fsh in which the length increment afects the weight of the fsh.Tis is in line with the opinion of [44] that the correlation coefcient value of the length-weight relationship is close to 1 showing that the increment of length will afect the increment of weight.Te determination coefcient (R 2 ) of wader abang fsh in this study is 0.9549, meaning that 95.49% of the fsh weight is Wader pari fsh has an R 2 value of 0.8989 meaning that 89.89% of the fsh weight is afected by its length and 10.11% by some other unknown factors.
Te condition factor is a very important factor used to evaluate fsh population and can be used as an indicator to estimate fsh body shape [54,55].Te mean value of the Fulton condition factor (KTL) of wader abang fsh is 1.07 and wader kepe fsh is 1.02, higher than the mean value of the condition factor of wader pari (0.77).Te shape of wader pari which is long and fat probably becomes a reason why its K value is low; this is to the fnding of [9], who stated that fsh with fat shapes tend to have a smaller condition factor.
Other than predicting the fsh's shape, condition factors can be used to describe a fsh's health status [56].Te mean value of relative condition factor (Kr) wader fsh from the Progo River is 95.435%-101.56%.According to [57], the relative condition factor is close to 100, showing that the fsh are in very good condition and there is a balance between prey and predator in the fsh habitat environment.Kr value of the three types of wader from the Progo River which is close to 100 indicates that the waters in the Progo River are still in good condition and stable enabling support for the growth of the wader fsh.Tis can be seen from the physicochemical parameters of waters in Progo River which are suitable for a fsh environment with an average pH of 8.8-8.9, temperature of 26-27 °C, dissolved oxygen (DO) of 7.4 mg/L, ammonia of 0.25 mg/L, nitrate of 5 mg/L, and phosphate of 0.5 mg/L (Table 7).

Conclusion
Based on the results of discriminant function analysis, all wader fsh from the Progo River used in this study were classifed into three species with the highest diferentiation character D4 (distance between the starting point of the upper tail fn and the starting point of the lower tail fn).Results also show that wader abang fsh have similar morphometric characteristics and DNA closer to wader kepe than to wader pari.
Te length-weight relationship of wader fsh from the Progo River shows that the growth pattern of wader abang and wader kepe is allometric positive, while that of wader pari is allometric negative.Te value of the condition factor for wader fsh from the Progo River indicates that the environmental habitat of wader fsh is excellent and stable so that it can support the fsh growth.

Table 2 :
Structure matrix of ordered correlation between measurement characteristics and functions for morphometric characteristics.
Notes: * gives a contribution to morphometric character diferences of wader fsh.

Table 3 :
Classifcation results of wader fsh from the Progo River based on morphometric characteristics.
Note. 96.6% of original grouped cases were correctly classifed.

Table 5 :
Te growth pattern of wader fsh in the Progo River is based on the length-weight relationship.

Table 6 :
Condition factor of Fulton (K) for wader fsh in the Progo River.

Table 7 :
Water quality of the Progo River.
afected by the length of the fsh while 4.51% is afected by some other unknown factors.Wader kepe has an R 2 value of 0.8086 meaning that the fsh weight is afected by the fsh length of 80.86% and 19.14% is afected by some other unknown factors.